PROJECT SUMMARY/ABSTRACT Current chemotherapy treatment for children with relapsed T-cell leukemia has a survival rate of less than 15%. Although children eligible for allogeneic hematopoietic stem cell transplant (HSCT) have a nearly 50% chance of survival, disease remission is necessary and is seldom achieved in relapsed T-cell leukemia. Immunotherapy using chimeric antigen receptors (CARs) is emerging as a powerful technology for targeted cancer therapeutics that can be utilized for the treatment of relapsed T-cell leukemia. CARs are predicted to provide specific targeting of tumor cells, unlike currently used anti-cancer agents, and could potentially provide children with relapsed T-cell disease the option to undergo allogeneic HSCT. Unfortunately, the clinical usefulness of CARs in numerous cancers has varied from producing curative results with virtually no side effects, to severe adverse events and even death. Clearly there is need to understand and improve upon this emerging technology. The antigen-binding domain of CARs, which generally consist of a single-chain variable fragment (scFv), is used to direct and activate genetically engineered cytotoxic immune cells. The expression of CARs, such as CD19-CARs, on T cells has demonstrated potent and successful immunotherapy for B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) and multiple myeloma in clinical trials. However, no successful CAR therapy for treating T-cell leukemia patients has been established, largely due to on-target but off-tumor effects. We propose to generate a CAR targeting the CD5 antigen on T cells and deliver this construct to innate immune ?? T cells and test the engineered cells in vitro and in vivo using a T-cell leukemia mouse model. It is proposed that ?? T cells are advantageous compared to ?? T cells because they do not form memory responses, which are predicted to be lethal when directed against T cells. Also, redirecting T cells to T-cell antigens causes CAR-modified T cells to self-activate and kill each other instead of the leukemic T cells. Therefore, we propose to knockout CD5 in ?? T cells using CRISPR/Cas9 technology. We designed and tested guide RNAs in conjunction with Cas9 using a single expression plasmid, which we showed is functional using a T-cell line. Additionally, our laboratory demonstrated the effectiveness of replacing the scFv domain of CARs with a variable lymphocyte receptor (VLR). VLRs are the main component of the lamprey adaptive immune system and are analogous to immunoglobulin, however, they have a fundamentally different structure resulting in novel binding partners inaccessible to scFvs. We have cloned an anti-CD5-VLR onto a CAR and confirmed its function on effector cells in vitro. We have also demonstrated lentiviral transduction of ?? T cells as a means of CAR delivery. Ultimately, our goal is to better understand how to engineer ?? T cells, but foremost to provide a treatment strategy to induce a state of remission for T-cell leukemia patients, allowing them to be eligible for HSCT. !